Synaptic activation of N-methyl- d-aspartate and non- N-methyl- d-aspartate receptors in the mossy fibre pathway in adult and immature rat cerebellar slices

Abstract

The participation of excitatory amino acid receptors in mossy fibre-granule cell synapses in lobule VIa of adult and immature rat cerebellar slices was investigated using an extracellular grease-gap technique. For the immature slices, the age selected (14 days after birth) was one at which the sensitivity of granule cells to exogenous N-methyl- d-aspartate is much higher than in the adult. The principal synaptic potentials observed after low-frequency electrical stimulation of the white matter resembled closely those found to be centred in the granule cell layer in field potential studies in the cat in vivo. They comprised a short latency negative potential, a slow negative wave and, in the adult, a further late negative wave. In the adult, with 1.2mM Mg 2+ in the perfusing solution, none of these potentials was significantly affected by the N-methyl- d-aspartate antagonist, 2-amino-5-phosphonovalerate, but they were all markedly inhibited by the broad spectrum antagonist, kynurenate, and, more potently, by the selective non- N-methyl- d-aspartate receptor blocker, 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline. After removal of Mg 2+, which has a blocking action on the ion channels associated with N-methyl- d-aspartate receptors, the size of all the potentials increased. The increase in the short latency potential was insensitive to 2-amino-5-phosphonovalerate but a component of the slow negative wave (and of the late negative wave) was reduced back to control levels by the antagonist. Application of 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (10μM) in Mg 2+-free solution revealed, in near isolation, a slow wave (latency to peak, 28 ms) which could be abolished by 2-amino-5-phosphonovalerate. In the immature slices, bathed in normal (Mg 2+-containing) medium, 2-amino-5-phosphonovalerate caused a small reduction in the short latency potential and inhibited a component of the slow negative wave which could, again, be observed in relative isolation after perfusion of 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline. Removal of Mg 2+ increased the amplitudes of the short latency potential and the slow negative wave in a manner which was sensitive to 2-amino-5-phosphonovalerate and increased the size of the slow, 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline-resistant wave. It is concluded that glutamate is likely to be the transmitter released by mossy fibres, at least those innervating lobule VIa. In the adult cerebellum, the transmitter acts on non- N-methyl- d-aspartate receptors to elicit the short latency potential, and on both N-methyl- d-aspartate and non- N-methyl- d-aspartate receptors in the synapses responsible for the slow negative wave, but the synaptic currents associated with the activation of the N-methyl- d-aspartate receptors are likely to be significant only under conditions where the block of the channels by Mg 2+ is reduced. In the immature cerebellum, N-methyl- d-aspartate receptors participate in both the short latency potential and the slow negative wave and their contribution to these potentials is significant even in the presence of Mg 2+. The involvement of N-methyl- d-aspartate receptors in the synaptic responses is likely to have implications both for the development of mossy fibre-granule cell synapses and for transmission through these synapses when they are mature.